Experimental Screening Protocols, Immunocytochemistry and Microscopy-based Imaging Techniques for Penium margaritaceum

Summary

This report describes the fundamental methods used to culture and experimentally manipulate the unicellular streptophyte alga, Penium margaritaceum. It also provides fundamental protocols of microscopy-based imaging, including live cell labeling with monoclonal antibodies and other fluorescent probes and scanning electron microscopy.

Abstract

The cell wall is the first component of signal reception/transduction for a plant cell during development and when responding to environmental abiotic and biotic stressors. The cell constantly monitors the integrity of its cell wall and modulates it in response to stress. Elucidating the specific structural and biochemical modulations occurring in the cell wall is a difficult task especially when employing multicellular plants and their organs/tissues. This is due to limits as to what can be resolved in an individual cell that is part of a complex multicellular network. The unicellular streptophyte alga, Penium margaritaceum, has recently been used in investigations of pectin dynamics, cell wall-based phenotypic plasticity and multiple aspects of algal cell biology. Its simple phenotype, distinct cell wall that has many components notably similar to land plant cell walls, and ease in immunocytochemical and experimental studies make it a powerful model organism in plant cell wall biology. The goal of this study is to provide the basic techniques for culturing, experimental manipulation and screening of applied stressors. Screening protocols for immunocytochemistry, confocal laser scanning microscopy imaging and scanning electron microscopy imaging of cell wall structure. Likewise, many of the described techniques may be modified for a wide array of other cell and molecular studies.

Introduction

The cell wall of a plant is a complex polymeric network that has multiple roles in the life of a plant cell¹. The integrity of the cell wall is constantly monitored by the cell during development and in response to environmental stress and, modulates in chemistry and structure accordingly. Penium margaritaceum is a unicellular green alga that has been recently used in studies of Streptophyte algae (Streptophyta, the group of green algae most closely related and ancestral to land plants²).

Over the past two decades, P. margaritaceum has been an important organism in investigat....

Protocol

NOTE: Penium margaritaceum is obtained at the Sammlung von Algenkulturen der Universität Göttingen - Culture Collection of Algae at Göttingen University, SAG; strain #2640.

1. Maintaining cultures

1. Maintain the alga in liquid Woods Hole medium (WH¹²) that may also be supplemented with soil water extract (i.e., 40 mL per L of medium). Maintain cultures at 20-25 °C with a 16 h:8 h light: dark cycle with 3.5 klux (74 µmol photons/m²/s) of cool white fluorescent light.
2. Prepare subcultures weekly and use cell cultures when they ar....

Results

The labeling of the cell wall of P. margaritaceum with anti-pectin mAbs (e.g., JIM5) reveals a network of calcium-complexed fibers and projections that form a regular pattern or lattice (Figure 1). The pectin is deposited in the cell center or isthmus, where it displaces older pectin toward the poles (Figure 2). Labeling with a different pectin antibody-like JIM7 highlights the initial secretion of high methyl-esterified pectin in a narrow band at the i.......

Discussion

P. margaritaceum is an efficacious organism for elucidating the dynamics of cell wall development and ECM secretion in plants and streptophyte algae. The main attributes include a unicellular habit and ease in culture maintenance and experimental manipulation, a primary cell wall with a distinct outer pectin lattice and other polymers, ease in live cell labeling with cell wall-directed mAbs that can be followed in time for subsequent developmental and/or experimental studies and the production of large amounts o.......

Materials

Name	Company	Catalog Number	Comments
1.5 mL microcent. Tubes	Fisher Scientific	01-549-740
12 welled microplate	Fisher Scientific	50-233-6077
22 x 22 mm coverslips	Fisher Scientific	12-541-016
45x 50 cm coverslips	Brain Research	4550-1.5D
Agar	Sigma Aldrich	A9414
anti-rat FITC	Sigma Aldrich	F6258
anti-rat TRITC	Sigma Aldrich	T4280
calcium chloride	Sigma Aldrich	C4981
Cambbridge stubs	EMS	75183-65
Fluoview CLSM	Evident	Fluoview 1200
JIM5	Kerafast	ELD004
JIM7	Kerafast	ELD005
Microcentrifuge	Fisher Scientific	13-100-675
Micropipetors	BioRad	1660499EDU
Penium margaritaceum	Sammlung von Algenkulturen der Universität Göttingen - Culture Collection of Algae at Göttingen University	2640
Polysphere kit	Polysciences	18336
SEM	ThermoFisher	Quattro SEM
sputter coater	EMS	Q150V
Vortex mixer	Fisher Scientific	02-215-414